Brain ischemia is characterized by a marked, rapid fall in pH, which is assumed to be injurious although through multiple or uncertain mechanisms. The recent discovery in brain of Acid Sensing Ion Channels (ASICs), which are ubiquitous and function physiologically in synaptic neurotransmission, offers a diffuse, membrane based, receptor gated ion channel system, which will respond to the pathologic pH fall in brain ischemia. These H+ receptor gated channels are Na+ channels, a portion of which is also Ca2+ permeable. Acid sensitivity and Ca2+ permeability suggest a role in ischemic brain injury. Using patch clamp techniques and Ca2+ imaging of native neurons in cortical cultures we show the pH sensitivity of these channels and acid induced Ca2+ uptake. Both the acid induced channel currents and Ca2+ uptake are greatly potentiated in the setting of modeled ischemia (Oxygen Glucose Deprivation-OGD or NaCN). Thus these channels respond to both acidosis and "ischemia" in a mutually potentiating manner. The channel current and Ca2+ uptake are glutamate independent, are inhibited by ASIC pharmacologic blockade and by specific blockade of the ASIC 1a channel subunit. Thus, our preliminary studies support new cellular and molecular mechanisms mediating ischemic-acidosis induced brain injury, which we offer to dissect with the following Specific Aims: Single cell recording and transfection of individual ASIC subunit cDNAs will show that: 1) Ca2+ permeable ASICs produce cell injury in modeled ischemia via a subunit specific mechanism. Using rat and mouse global ischemia models and ASIC1a & ASIC2a knockout mice, we propose that: 2) Blockade of ASICs protects against ischemic brain injury in vivo. With molecular techniques including site directed mutagenesis we will describe endogenous Zn2+ modulation of Ca2+permeable ASICs: 3) Characterize high-affinity Zn2+ modulation of ASIC currents in acidosis and ischemia -induced cell injury. These experiments will describe new, glutamate independent, mechanisms of ischemic brain injury and the central role of ischemic-acidosis. ASIC blockade will offer new and potent potential therapy for stroke.